Project Summary The overall goal of this proposal is to determine the mechanism by which Insulin Receptor Substrate 2 (IRS2) promotes invasion and its role in breast cancer. IRS2 is a cytoplasmic adaptor protein that is a key signaling effector of the insulin (IR) and insulin-like growth factor-1 (IGF1R) receptors, both of which have been implicated in breast cancer. Mouse mammary tumors that lack Irs2 are significantly diminished in their ability to metastasize and tumors with elevated Irs2 expression have enhanced tumor growth and metastatic potential. Work from the applicant?s lab has established that IRS2 promotes invasion, an early step in the dissemination of metastatic cells to secondary organs. The significance of IRS2 promoting invasion is heightened by the applicant?s recent discovery that IRS2 is recurrently mutated in pleomorphic invasive lobular carcinoma (PILC), an aggressive, metastatic breast cancer subtype. Importantly, the mechanism by which IRS2 integrates upstream signals to mediate its functional outcomes remains unknown. Determining how IRS2 regulates invasion requires an understanding of its structure, and how this structure determines function. The applicant?s preliminary data establish that the ability of IRS2 to promote invasion is dependent upon upstream IGF1R/IR activation and the recruitment and activation of PI3K. In addition, they identified a 174-amino acid region within the IRS2 C-terminal tail that is required for invasion. Importantly, this region is not required for the IRS2-dependent regulation of glucose uptake, revealing that these two functions of IRS2 are independently regulated. Essential interactions likely occur within this region given that it acts in a dominant negative manner to inhibit invasion. To investigate the hypothesis that the structure of IRS2 is dynamically altered by upstream stimuli that promote invasion to facilitate binding of essential downstream signaling effectors the applicant will: 1) Define the structural basis for IRS2-mediated invasion and signaling. The hypothesis that specific sequences within IRS2 participate in dynamic intramolecular interactions that alter protein conformation and signaling to promote invasion will be examined; 2) Investigate IRS2 interacting partners and their role in promoting invasion. The hypothesis that intramolecular interactions within IRS2 lead to the formation of ?disordered domain? loops that assemble distinct signaling sub-complexes to mediate functional outcomes, and that one of these binding proteins is the serine threonine kinase BMP2K, will be examined; 3) Establish the role of IRS2-dependent invasion in breast cancer progression in vivo. The hypothesis that selective targeting of IRS2-dependent invasion will reveal an essential role for this specific function in breast cancer progression will be examined. The contribution of IRS2 mutations to PILC progression will also be examined.